This is a multi-user application to purchase an inverted, widefield fluorescence microscope with high sensitivity and resolution for 3D image acquisition, deconvolution, and live cell time lapse and fluorescence recovery after photobleaching (FRAP). High sensitivity and rapid data acquisition for 3D time lapse will be achieved by using a 10 MHz Cascade II EMCCD camera combined with deconvolution software. The instrument will provide flexibility for use in imaging live and fixed mammalian and yeast cells as well as tissue slices for a diverse group of users. The Fuller laboratory is studying the molecular basis of trafficking between late Golgi and endosomal compartments in yeast. This instrument will be used to measure the dynamics of transport vesicle formation and consumption and of the association of trafficking proteins with organelles using, as probes, functional fluorescent-protein fusions to endogenous proteins. The Engelke lab is examining the three-dimensional organization of nuclear genomes in the budding yeast, Saccharomyces cerevisiae, and in murine and human cells. Information retrieval in nuclei involves complex and spatially controlled positioning of genes, and this positioning is dynamic in response to metabolic and developmental signals. The mechanism is being investigated for clustering of RNA polymerase III promoters as a means of genome condensation and organization. The Collins lab is focused on the how the human immunodeficiency virus (HIV-1) evades the immune system in establishing a persistent infection. Collins and coworkers have shown that one way this is accomplished is via the activity of the HIV- 1 Nef protein, which disrupts antigen presentation by altering the intracellular trafficking of MHC- I. 3D and 4D imaging will be used to map the effects of on MHC-I, other membrane proteins and coat and adaptor proteins in primary T-cells and T-cell lines. Experiments will evaluate effects of mutations in nef interaction sites and knockdowns of cellular transport factors. The Banerjee lab is studying remodeling of the extra- and intra-cellular redox potential during effector T cell activation and its modulation by regulatory T cells. They use fluorescent sensors to track changes in the redox status of exofacial proteins and for the direct readout of the intracellular glutathione: glutathione disulfide redox potential. Minor users of the instrument (and areas of interest) will include Professors Carol Fierke (compartmentation of zinc metabolism), Tom Kerppola (cellular ubiquitination pathways, bimolecular fluorescence complementation), Stephen Ragsdale (heme oxygenase-2 interactions), Anne Vojtek (signaling mechanisms regulating neuronal morphogenesis), Aaron Goldstrohm (RNA stability and degradation).